Runx1-CBFbeta is a heterodimeric transcription factor required for the emergence of hematopoietic stem cells in the embryo. During postnatal life mutations in the RUNX1 and CBFB genes in a hematopoietic stem cell or committed progenitor contributes to the formation of human leukemias. The RUNX1 (AML1) gene is disrupted by about a dozen different chromosomal translocations, including the t(8;21), t(12;21), and t(3;21) in acute myeloid and pediatric lymphoblastic leukemias, and in therapy related leukemias and myelodysplasias, respectively. The CBFB gene is rearranged in acute myeloid leukemias by the inv(16). Together, the RUNX1 and CBFB genes are rearranged in approximately one quarter of all acute myeloid and lymphoblastic leukemias. Here we propose to examine the requirement for the Runx1-CBFbeta heterodimer throughout normal hematopoietic development in mice. We will determine whether Runx1-CBFbeta function in a specialized "hemogenic endothelium" is necessary and sufficient in order to produce the first hematopoietic stem cells. We will begin to identify the signals that specify the first hematopoietic stem cells by characterizing the cis-acting sequences that regulate Runx1 expression in the embryo. Finally we will examine the requirement for continued Runx1-CBFbeta function during later stages of postnatal hematopoiesis.